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Waters 474 Scanning Fluorescence Detector

Operator’s Guide

Waters 474 Scanning Fluorescence Detector Operator’s Guide 34 Maple Street Milford, MA 01757 WAT047467, Revision 3
Waters 474 Scanning Fluorescence Detector Operator’s Guide 34 Maple Street Milford, MA 01757 WAT047467, Revision 3
Waters 474 Scanning Fluorescence Detector Operator’s Guide 34 Maple Street Milford, MA 01757 WAT047467, Revision 3

34 Maple Street Milford, MA 01757

NOTICE

The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation. Waters Corporation assumes no responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual.

1996, 2000 WATERS CORPORATION. PRINTED IN THE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED. THIS BOOK OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

Millennium and Waters are registered trademarks of Waters Corporation.

All other trademarks are the sole property of their respective owners.

are registered trademarks of Waters Corporation. All other trademarks are the sole property of their respective

Attention: When you use the instrument, follow generally accepted procedures for quality control and methods development.Attention:

If you observe a change in the retention of a particular compound, in the resolution between two compounds, or in peak shape, immediately take steps to determine the reason for the changes. Until you determine the cause of a change, do not rely upon the results of the separations.

Caution: For continued protection against fire hazard, replace fuses with those of the same type and For continued protection against fire hazard, replace fuses with those of the same type and rating.

Attention: Changes or modifications to this unit not expressly approved by the party responsible for compliance Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment.

Note: The Installation Category (Overvoltage Category) for this instrument is Level II. The Level II category pertains to equipment that receives its electrical power from a local level, such as an electrical wall outlet.

Symbols Used on 474 Detector

Direct current

Direct current

Alternating current

Alternating current

Protective conductor terminal

Protective conductor terminal

Frame or chassis terminal

Frame or chassis terminal

Caution, risk of electric shock

Caution, risk of electric shock

Caution or refer to manual

Caution or refer to manual

UV
UV

Ultraviolet light

Hot surfaces

Hot surfaces

474 Scanning Fluorescence Detector Information

Intended Use

The Waters 474 Scanning Fluorescence Detector can be used for in-vitro diagnostic testing to analyze many compounds, including diagnostic indicators and therapeutically monitored compounds. When developing methods, follow the “Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory,” American Journal of Medical Technology, 44, 1, pages 30 – 37 (1978). This protocol covers good operating procedures and techniques necessary to validate system and method performance.

Biological Hazard

When analyzing physiological fluids, take all necessary precautions and treat all specimens as potentially infectious. Precautions are outlined in “CDC Guidelines on Specimen Handling,” CDC – NIH Manual, 1984.

Calibration

Follow acceptable methods of calibration with pure standards to calibrate methods. Use a minimum of five standards to generate the standard curve. Concentration range should cover the entire range of quality control samples, typical specimens, and atypical specimens.

Quality Control

Routinely run three quality-control samples. Quality-control samples should represent subnormal, normal, and above-normal levels of a compound. Ensure that quality-control sample results are within an acceptable range, and evaluate precision form day to day and run to run. Data collected when quality control samples are out of range may not be valid. Do not report this data before ensuring that chromatographic system performance is acceptable.

Table of Contents

How to Use This Guide

19

Chapter 1 Introduction and Theory of Operation

22

1.1 Introduction

22

1.2 Waters 474 Detector Features

23

1.3 Fluorescence Theory of Operation

24

1.3.1 Excitation Sources

24

1.3.2 Filtering

25

1.3.3 Exciting the Sample

26

1.3.4 Measuring Fluorescence

26

1.3.5 Scanning

27

1.3.6 References

27

1.4 Principle of Operation

28

1.4.1 Optics Bench

28

1.4.2 Control System

30

Chapter 2 Installing the 474 Detector

32

2.1 Site Selection and Power Requirements

32

2.2 Unpacking and Inspection

33

2.3 Voltage Selection and Fuse Installation

35

2.4 Making Fluid Connections

36

2.5 Making Electrical Power Connections

39

2.5.1

Making AC Power Connections

39

2.6

Making Connections to Other Devices

40

2.6.1 Analog Connector Overview

40

2.6.2 Connecting the 474 to the Millennium Data System

43

2.6.3 Connecting the 474 to the Waters 2690 Separations Module

48

2.6.4 Connecting the 474 to the Waters 600 Series Controller

53

2.6.5 Connecting the 474 to a Waters 745/745B/746 Data Module

58

2.6.6 Connecting the 474 to a Chart Recorder

59

2.6.7 Connecting the 474 to the Waters 717plus

60

2.6.8 Connecting the 474 to the Carbamate System

62

2.6.9 Connecting the 474 to the AccQTag System

65

2.6.10 Connecting a Voltmeter to the Excitation Out Terminals

67

Chapter 3 Using the 474 Detector

68

3.1 Powering On

68

3.2 Using the Front Panel

69

3.3 Key Descriptions

71

3.4 Operating Parameters

77

 

3.4.1 Displaying and Changing Parameter Settings

78

3.4.2 Excitation Wavelength

79

3.4.3 Emission Wavelength

79

3.4.4

Gain

79

3.4.5 Attenuation

80

3.4.6 Response

80

3.5 Special Parameters

81

3.5.1 Program Number

82

3.5.2 Diagnostics

82

3.5.3 Energy Output

83

3.5.4 Lamp Off Timer

83

3.5.5 Signal Filter

84

3.5.6 Auto Zero Enable

87

3.5.7 Offset

89

3.5.8 Polarity

90

3.5.9 Lamp Hours

91

3.5.10 Bandwidth

92

3.5.11 Wavelength Range

94

3.5.12 Temperature Correction

95

3.6 Using External Functions

95

3.6.1 Excitation Energy Out

96

3.6.2 Mark Out

96

3.6.3 Cell Leak

96

3.6.4 Mark In

97

3.6.5 Auto Zero In

97

3.6.6 Program Reset

97

3.7 Calibration Check

98

3.7.1

Excitation Wavelength Accuracy (Raman Band of Water )Test

98

3.7.2

Signal-to-Noise Test

102

3.8

Powering Off

103

Chapter 4 Programming the 474 Detector

105

4.1 Using the Program Mode

105

4.2 Writing a Multi-Step Time Program

107

 

4.2.1 Setting the Initial Conditions of a Time Program

109

4.2.2 Setting Steps in a Time Program

111

4.2.3 Saving the Time Program

113

4.2.4 Example Time Program

114

4.3 Running a Time Program

119

 

4.3.1 Manual Time Program Start

119

4.3.2 External Time Program Start

120

4.4 Modifying a Time Program

121

 

4.4.1 Accessing the Program Number

121

4.4.2 Modifying a Program Step

122

4.4.3 Adding a Program Step

123

4.4.4 Deleting a Program Step

124

4.4.5 Deleting a Group of Steps

124

4.5 Performing a Wavelength Scan in a Time Program

125

 

4.5.1 Storing a Spectrum

126

4.5.2 Entering the Spectrum Parameters

128

Chapter 5 Using the Scan Mode

129

5.1 Scan Parameters

130

5.2 Performing Spectral Scans

132

5.2.1 Manual Emission Spectrum Scans

133

5.2.2 Manual Excitation Spectrum Scans

134

5.3 Spectrum Output

136

5.3.1 Outputting Emission Spectra

137

5.3.2 Outputting Excitation Spectra

139

5.4 Difference Spectra

142

5.4.1 Emission Difference Spectrum

143

5.4.2 Excitation Difference Spectra

146

Chapter 6 Maintenance Procedures

149

6.1 Routine Maintenance

151

6.2 Cleaning the Ventilation Filters

151

6.3 Cleaning the Flow Cell

152

6.4 Replacing the Flow Cell

153

6.4.1 Removing the Flow Cell Cassette

153

6.4.2 Disassembling the Flow Cell Cassette

154

6.4.3 Reassembling the Flow Cell

157

6.4.4 Reassembling the Flow Cell Cassette

158

6.4.5 Reinstalling the Flow Cell Cassette

159

6.5 Changing the Lamp

159

6.5.1

Removing the Lamp Housing

160

6.5.2

Replacing the Lamp Housing

164

 

6.5.3

Adjusting the Lamp

164

6.6

Replacing AC Fuses

166

Chapter 7 Error Messages and Diagnostics

167

7.1 Error Messages

167

 

7.1.1 Power-On Diagnostic Tests

167

7.1.2 Operating Errors

168

7.2 Diagnostics

 

170

Chapter 8 Troubleshooting

171

8.1 General Troubleshooting Hints

172

8.2 Hardware Troubleshooting

173

Appendix A Specifications

 

175

Appendix B Spare Parts and Accessories

178

B.1 Using the Optional Cuvette Cell

179

B.2 Using the Optional Optical Filters

181

2.3

Optional Photomultiplier Tubes

185

Appendix C Warranty Information

187

C.1

Limited Express Warranty

187

C.2

Shipments, Damages, Claims, Returns

191

Index

192

List of Figures

1-1

Waters 474 Scanning Fluorescence Detector

22

1-2

The 474 Detector Optics Bench

29

1-3

Block Diagram of the 474 Detector

31

2-1

Voltage Selection Switch and Fuse Holders

35

2-2

Fluid Connections

37

2-3

Ferrule and Compression Screw Assembly

38

2-4

Rear Panel Electrical Connectors

39

2-5

Analog Connector

41

2-6

Bus SAT/IN Module (Front Panel)

44

2-7

Bus SAT/IN to Bus LAC/E Connections

45

2-8

Bus SAT/IN Module Connections

46

2-9

Waters 2690 Separations Module Auto Zero Connections

49

2-10

Waters 2690 Separations Module Chart Mark Connections

50

2-11

Waters 2690 Separations Module Method Start Connections

51

2-12

Waters 2690 Separations Module Cell Leak Connections

52

2-13

Waters 600 Series Controller Auto Zero Connections

54

2-14

Waters 600 Series Controller Chart Mark Connections

55

2-15

Waters 600 Series Controller Connections

56

2-16

Waters 600 Series Controller Cell Leak Connections

57

2-17

Waters 745/745B/746 Connections

58

2-18

Chart Recorder Connections

60

2-19

Waters 717plus Auto Zero Connections

61

2-20

Waters Carbamate System Chart Mark Connections

63

2-21

Waters Carbamate System Inject Start Connections

64

2-22

AccQTag System Chart Mark Connections

66

2-23

Waters AccQTag System Connections

67

3-1

474 Detector Front Panel

70

3-2

Typical Display in Normal Mode

71

3-3

Keypad Layout

73

3-4

Typical Display in the Normal Mode

78

3-5

Typical Energy Output Display

83

3-6

Lamp Off Timer Selection Screen

84

3-7

Display Screen After Lamp Timer Has Elapsed

84

3-8

Filter Selection Screen

85

3-9

Effect of Different Settings in the RC Filter Mode

86

3-10

Effect of Different Settings in the Digital Filter Mode

87

3-11

Auto Zero Selection Screen

89

3-12

Offset Selection Screen

90

3-13

Effect of Changing the Polarity Parameter

91

3-14

Polarity Selection Screen

91

3-15

Lamp Hours Display Screen

92

3-16

Bandwidth Selection Screen

93

3-17

Wavelength Range Selection Screen

94

3-18

Temperature Correction Display Screen

95

3-19

Error Message for a Cell Leak

97

3-20

Entering the Emission Spectrum Memory Location

99

3-21

Entering the Excitation Wavelength for an Emission Spectrum

99

3-22

Prompt to Begin an Emission Spectrum Scan

99

3-23

Starting an Emission Spectrum Output

100

3-24

Entering the Emission Spectrum Memory Location

100

3-25

Entering the Start and End Wavelengths in the Emission

Spectrum

100

3-26

Prompt to Start Output of a Spectrum

101

3-27

Typical Raman Band Test Results

102

3-28

Measuring the Maximum Pen Deflection

103

4-1

Typical Time Screen in the Program Mode

106

4-2

Typical Fluorescence Intensity Screen in the Program Mode

106

4-3

Flowchart for Writing a Time Program

108

4-4

Typical Initial Parameter Settings

109

4-5

First Step of a New Time Program

111

4-6

Typical First Step of an Existing Time Program

111

4-7

Typical Empty Program Step

112

4-8

First Line of the Program Menu

112

4-9

Initial Conditions of the Example Time Program

115

4-10

Step 1 of the Example Time Program

116

4-11

Step 2 of the Example Time Program

117

4-12

Step 3 of the Example Time Program

118

4-13

Step 4 of the Example Time Program

119

4-14

Fluorescence Intensity Screen at the End of a Program Run

120

4-15

Editing the Parameters in a Time Program

122

4-16

Typical Spectrum Output by the 474 Detector

125

4-17

Conditions for Storing a Spectrum

126

4-18

Typical Values for Storing an Emission Spectrum

127

5-1

Initial Display in the Scan Mode

129

5-2

Selecting the Scan Parameters

131

5-3

Entering the Scan Parameters

131

5-4

Entering the Emission Spectrum Memory Location

133

5-5

Entering the Excitation Wavelength for an Emission Spectrum

134

5-6

Prompt to Begin an Emission Spectrum Scan

134

5-7

Starting an Excitation Spectrum Scan

135

5-8

Entering the Excitation Spectrum Memory Location

135

5-9

Entering the Emission Wavelength for an Excitation Spectrum

135

5-10

Prompt to Begin an Excitation Spectrum Scan

136

5-11

Starting an Emission Spectrum Output

137

5-12

Entering the Emission Spectrum Memory Location

138

5-13

Entering the Start and End Wavelengths in the Emission

Spectrum

138

5-14

Prompt to Start Output of a Spectrum

139

5-15

Typical Display During Output of a Spectrum

139

5-16

Starting an Excitation Spectrum Output

140

5-17

Entering the Excitation Spectrum Memory Location

140

5-18

Entering the Start and End Wavelengths in the Excitation

Spectrum

140

5-19

Prompt to Start Output of a Spectrum

141

5-20

Typical Display During Output of a Spectrum

141

5-21

Starting an Emission Difference Spectrum Output

143

5-22

Entering the Memory Locations for an Emission Difference

Spectrum

143

5-23

Incorrect Wavelengths for a Difference Spectrum

144

5-24

Entering the Start and End Wavelengths for an Emission

Difference Spectrum

144

5-25

Prompt to Begin an Emission Difference Spectrum

145

5-26

Typical Display During Output of an Emission Difference

Spectrum

145

5-27

Starting an Excitation Difference Spectrum Output

146

5-28

Entering the Memory Locations for an Excitation Difference

Spectrum

146

5-29

Incorrect Wavelengths for a Difference Spectrum

147

5-30

Entering the Start and End Wavelengths for an Excitation

Difference Spectrum

147

5-31

Prompt to Begin an Excitation Difference Spectrum

148

5-32

Typical Display During Output of an Excitation Difference

Spectrum

148

6-1

Removing the Flow Cell Cassette

154

6-2

Flow Cell Cassette

155

6-3

Exploded View of Flow Cell Parts

156

6-4

Position of Cell Mask

158

6-5

Accessing the Lamp Housing

161

6-6

Moving the Printed Circuit Board Panel

162

6-7

Removing the Screws That Secure the Fan Access Door

162

6-8

Removing the Securing Screws on the Lamp Housing

163

6-9

Horizontal Adjustment of the Lamp

165

7-1

Error Message for Fluid Leak

169

7-2

Auto Zero Error Message

169

B-1

Installing the Optional Cuvette Cell Cassette

180

B-2

Characteristics of Excitation Filters

182

B-3

Characteristics of Emission Filters

183

B-4

Installing Optical Filters on the Flow Cell

184

B-5

Sensitivity of Optional Photomultiplier Tubes

186

List of Tables

2-1

Installation Site Requirements

32

2-2

Voltage and Fuse Selection

33

2-3

Analog Connector Terminal Descriptions

42

3-1

Display Parameters

71

3-2

Key Descriptions

74

3-3

Parameters, Ranges, and Keys

78

3-4

Filter Modes and Response Settings

80

3-5

Special Parameter Menu

81

3-6

Effects of Manual and Automatic Auto Zero Settings

88

4-1

Program Menu

113

4-2

Example Program

114

5-1

Scan Menu

129

5-2

Scan Parameters

130

6-1

AC Fuses

166

7-1

Power-On Diagnostic Tests

168

7-2

Possible Causes of the Auto Zero Error Message

170

8-1

General System Troubleshooting

173

A-1

Operational Specifications

175

B-1

Spare Parts and Accessories

178

B-2

Optional Optical Filter Characteristics

181

B-3

Photomultiplier Tube Type Numbers

185

C-1

Waters 474 Scanning Fluorescence Detector Warranty Periods

190

How to Use This Guide

Purpose of This Guide

The Waters 474 Scanning Fluorescence Detector Operators Guide details the procedures for unpacking, installing, operating, maintaining, and troubleshooting the 474. It also includes appendixes describing specifications, spare parts and accessories, and warranty information.

Audience

This guide is intended for use by personnel who need to install, operate, maintain, or troubleshoot the 474 detector.

This guide assumes that you understand the principles of chromatography.

Structure of This Guide

The Waters 474 Scanning Fluorescence Detector Operators Guide is divided into eight chapters. Each chapter page is marked with a chapter-specific tab and a footer to help you quickly find information. Each appendix page is identified in a similar manner.

The following table describes the material covered in each chapter.

Chapter/Appendix

Content

Chapter 1, Introduction and Theory of Operation

Presents a description of the 474 Scanning Fluorescence Detector, including the theory of operation and its features.

Chapter 2, Installing the 474 Detector

Describes how to install the 474 detector, including making all electrical and fluidic connections.

Chapter 3, Using the 474 Detector

Provides the power-on and power-off procedures, and describes how to set and change operating parameters.

Chapter 4, Programming the 474 Detector

Describes how to write a time program.

Chapter 5, Using the Scan Mode

Describes how to make spectral scans and perform spectral manipulation.

Chapter/Appendix

Content

Chapter 6, Maintenance Procedures

Covers routine maintenance procedures.

Chapter 7, Error Messages and Diagnostics

Explains 474 detector error messages and start-up diagnostics.

Chapter 8, Troubleshooting

Describes troubleshooting procedures for the 474 detector, including troubleshooting decision trees and symptom-cause-solution tables.

Appendix A, Specifications

Describes the specifications of the 474 detector.

Appendix B, Spare Parts and Accessories

Lists recommended and optional spare parts and procedures for installing options.

Appendix C, Warranty Information

Provides warranty and service information.

Related AdobeAcrobat Reader Documentation

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Printing From This Electronic Document

Adobe Acrobat Reader lets you easily print pages, pages ranges, or the entire electronic document by selecting Print from the File menu. For optimum print quantity, Waters recommends that you specify a Postscript printer driver for your printer. Ideally, use a printer that supports 600 dpi print resolution.

Conventions Used in This Guide

This guide uses the following conventions to make text easier to understand.

Purple Text indicates user action. For example:

Press 0, then press Enter for the remaining fields.

Italic text denotes new or important words, and is also used for emphasis. For example:

An instrument method tells the software how to acquire data.

Underlined, Blue Color text indicates hypertext cross-references to a specific chapter, section, subsection, or sidehead. Clicking this topic using the hand symbol automatically brings you to this topic within the electronic document. Right-clicking and selecting Go Back from the popup context menu brings you back to the originating topic. For example:

Remove the standard flow cell cassette as described in Section 6.4.1, Removing the Flow Cell Cassette.

Notes, Attentions, and Cautions

Notes call out information that is important to the operator. For example:

Note: Record your results before you proceed to the next step.

Attentions provide information about preventing possible damage to the system or equipment. For example:

Attention: To avoid damaging the detector flow cell, do not touch the flow cell Attention: window. window.

Cautions provide information essential to the safety of the operator. For example:

Caution: To avoid chemical or electrical hazards, always observe safe laboratory practices when operating the system.Caution:

Caution: To avoid the possibility of electrical shock and possible injury, always turn Caution: off the detector and unplug the power cord before performing maintenance procedures. off the detector and unplug the power cord before performing maintenance procedures.

Caution: To avoid the possibility of burns, turn off the lamp at least 30 minutes before To avoid the possibility of burns, turn off the lamp at least 30 minutes before removing it for replacement or adjustment.

1

1

Introduction and Theory of

Operation

This chapter introduces you to the Waters® 474 Scanning Fluorescence Detector. It provides a summary of the features and a brief description of the theory and principle of operation.

1.1 Introduction

The Waters 474 Scanning Fluorescence Detector (Figure 1-1) is a high-performance unit that provides a
The Waters 474 Scanning Fluorescence Detector (Figure 1-1) is a high-performance unit
that provides a high level of sensitivity for spectrofluorometric analysis.
Waters™ 474
Scanning Fluorescence Detector
out
16µL Flow Cell
!
pressure
1000 max
kPa (145
psi)
in
O
I
TP01140

Figure 1-1 Waters 474 Scanning Fluorescence Detector

1.2 Waters 474 Detector Features

The Waters 474 Scanning Fluorescence Detector includes:

An optical design that maximizes sensitivity

Microprocessor control for greater versatility and reliability

An emission wavelength sweep function so you can obtain an emission spectrum

An excitation wavelength sweep function so you can obtain an excitation spectrum

An automatic second-order filter to enhance applications in the visible wavelength range

Optional optical filters that can be installed on the flow cell for specialized measurements

Quartz flow cell or cuvette cell, permitting measurement of ultraviolet fluorescence

Nine-stage photomultiplier tube for amplifying the emission radiation

Photodetector to compensate for excitation fluctuations throughout a scan

An RS-232 port to allow connectivity to the Millennium 32 data system

1

There are three critical optical design features in the 474 detector:

A highly energy-efficient optics system that does not use lenses, minimizing reflection loss

An optics system that uses holographic concave diffraction gratings and aspherical mirrors to provide highly efficient monochromators in a compact package

A 150 W Xenon lamp that provides greater light energy for excitation

There are five critical microprocessor-based features:

Operating parameters that are set using the keypad

A multi-line liquid crystal display (LCD) that provides information on the status of the 474 detector

Self-diagnostic messages that are displayed on the LCD

Digital processing that controls the entire signal processing system, assuring higher reliability and operational stability

An auto-zero feature that provides a wide range of offset adjustment for the analog output

1

1.3 Fluorescence Theory of Operation

This section describes the general theory of operation of fluorescence detection. The process of fluorescence detection involves several components and processes:

An excitation source

Filtering the source light

Exciting the sample with filtered light

Collecting and filtering the emitted fluorescence

Measuring the emitted fluorescence

Amplifying the emitted signal

Overview

When certain molecules or atoms are exposed to an energy source such as high-intensity light, the molecules or atoms absorb the light energy and enter into an excited state. As a molecule or atom moves from this excited state back to its normal state, some of the absorbed energy is released in the form of a photon. This process is called fluorescence.

Fluorescence is a type of luminescence. The atoms and molecules in different elements and compounds emit different levels of fluorescence when exposed to the same energy levels.

The scanning fluorescence detector illuminates a sample with a narrow band of high-intensity light from a carefully controlled light source. The detector then measures the low levels of fluorescence emitted from the sample. The emitted light is filtered, amplified, and converted to electrical signals that can be recorded and analyzed.

Applications

Fluorescence detectors are widely used for detecting:

Trace metals

Traces of organic material

Trace elements in biological systems

1.3.1 Excitation Sources

The typical energy source used for fluorescence detection is a lamp that provides an intense, stable spectrum of light in the UV and visible range. The fluorescence intensity is directly related to the intensity of the excitation spectrum, so high-sensitivity detectors use the most intense excitation source available.

Types of Light Sources

Common excitation light sources include the following:

Vapor lamps: Mercury, Cadmium, or Zinc

Arc lamps: Deuterium or Xenon

Of these light sources, the vapor lamps provide high-intensity, narrow-band outputs in the UV and visible ranges. The arc lamps provide a wider, lower intensity spectrum.

Among the arc lamps, the Xenon lamp provides the widest spectrum, making it the excitation source of choice for general-purpose fluorescence detectors.

1.3.2 Filtering

1

You select the desired excitation wavelength using some form of filtering. Two common methods of filtering the source light are typically used in modern detectors:

Optical filters

Monochromators

Optical Filters

Optical filters are generally low in cost and simple to use. They transmit a wide range of wavelengths, and, therefore, do not provide the degree of selectivity that a monochromator does.

Short-wavelength cut-off filters transmit light at wavelengths shorter than their rated value. Long- wavelength cut-off filters transmit wavelengths longer than their rated value. The rated value of a filter is defined as the wavelength at which 50 percent of the peak transmission occurs. This value is fixed; to change the excitation wavelength when you are using filters, the filter must be changed.

Monochromators

A

monochromator is a device that can be adjusted to select wavelengths over a wide range

of

the spectrum. A grating monochromator uses a diffraction grating that at any one setting,

passes only a small range, or bandwidth of wavelengths. By moving the grating, you can select wavelengths within the desired range of wavelengths. Relative to filters, the grating provides the following benefits:

A wider range of wavelengths can be selected

A smaller bandpass of wavelengths (greater selectivity)

Scans of the spectrum

1

A grating monochromator also passes fractions, or orders, of the selected wavelength. For

example, if the monochromator is set to pass light energy at 600 nm, it also passes energy

at the second-order wavelength of 300 nm. A long-pass filter can be used to absorb the

higher-order energy produced by a monochromator.

Just as the excitation, or source energy, is selected by a monochromator, the emission, or radiated energy, can be selected. This permits precise selection of emission wavelengths. Detectors with excitation and emission monochromators can perform scans by holding one monochromator at a constant setting while varying the setting on the other. This type

of operation is necessary when you are evaluating mixtures or analyzing the chemical

structure of a substance.

1.3.3 Exciting the Sample

The broad band of high-intensity light from the lamp passes through a filter or monochromator, which selects a narrow band of wavelengths. This narrow band of light is directed onto the flow cell (or the optional cuvette cell) where it excites the analytes as they pass through the cell.

Flow Cell

The flow cell is a glass or quartz cell designed to minimize the amount of stray light that could affect the measurement, and to maximize the fluorescence signal. The sample compartment is arranged so that the fluorescence energy is collected at an angle perpendicular to the excitation (lamp) beam.

1.3.4 Measuring Fluorescence

To measure the fluorescence from the flow cell, the detector must balance the need for high selectivity (to distinguish narrow fluorescence peaks) with the need for high sensitivity (to measure low fluorescence intensities).

As you increase the selectivity, you decrease the bandpass, or range of fluorescence wavelengths that pass through the filter. The narrower the filter band, the less light of fluorescence passes through it, and therefore, the less sensitive the measurement. If higher sensitivity is needed, more fluorescence must pass through the filter, that is, the bandpass must increase. This correspondingly decreases the ability of the filter to detect narrow fluorescence peaks.

Emission Monochromator

An emission monochromator provides the best means of balancing between sensitivity and selectivity. If greater selectivity is desired and the fluorescence level is sufficiently high, some detectors allow you to select a narrower slit. This can be useful when you need to eliminate or reduce the effects of coeluting compounds or impurities.

Photomultiplier Tube (PMT)

The fluorescence intensity is typically very low, especially if you are dealing with low levels of analyte. A sensitive photomultiplier tube (PMT) converts the fluorescence energy (in the UV, visible, or IR ranges) to an electric current. As its name implies, the PMT amplifies the low-level fluorescence signal.

1.3.5 Scanning

1

Detectors equipped with excitation and emission monochromators can easily scan a range of excitation or emission wavelengths. Changing the wavelength involves changing the monochromator setting. During a scan, the setting on one monochromator is held constant while the other monochromator scans a range of wavelengths.

As the excitation wavelength changes in a scan, the intensity from the source lamp tends to fluctuate. To compensate for these fluctuations, a beam splitter diverts a portion of the excitation energy to a photodetector. The photodetector monitors the fluctuations caused by changes in the excitation wavelength and provides a reference signal to the PMT. The 474 detector compensates for the changes in the fluorescence intensity caused by the fluctuations in excitation energy by using the ratio of the fluorescence and reference signals.

1.3.6 References

Refer to the following texts for additional information on fluorescence detection:

N. Ichinose, G. Schwedt, F.M. Schnepel, and K. Adachi, Fluorometric Analysis in Biomedical Chemistry, Chapter 5, Wiley-Interscience: New York, 1991.

Detectors for Liquid Chromatography, E.S. Yeung, ed., Chapter 5, Wiley: New York,1986.

W.R. Seitz, in Treatise on Analytical Chemistry, 2nd Ed., P.J. Elving, E.J. Meehan, I.M. Kolthoff, Eds., Part I, Vol. 7, Chapter 4, Wiley: New York, 1981.

J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum: New York, 1983.

S.G. Schulman, Fluorescence and Phosphorescence Spectroscopy: Physicochemical Principles and Practice, Pergamon Press: New York, 1977.

J.D. Winefordner, S.G. Schulman, and T.C. OHaver, Luminescence Spectroscopy in Analytical Chemistry, Wiley-Interscience: New York, 1972.

1

1.4 Principle of Operation

The Waters 474 Scanning Fluorescence Detector consist of two basic internal systems:

An optical system, often called the optics bench

A control system

1.4.1 Optics Bench

The optical bench consists of the following components:

Light source

Excitation monochromator

Beam splitter

Flow cell

Emission collector optics

Emission monochromator

Photomultiplier tube

Figure 1-2 shows these components and the light path through the 474 detector.

Figure 1-2 The 474 Detector Optics Bench The optics bench operates as follows: 1 1.

Figure 1-2 The 474 Detector Optics Bench

The optics bench operates as follows:

1

1. Energy from the light source is concentrated and directed toward the entrance slit of the excitation monochromator by an elliptical mirror.

2. The excitation monochromator diffracts the broadband source light and projects it onto a second slit to obtain the excitation spectrum. This spectrum is directed to the flow cell by the illumination optics.

3. A beam splitter diverts part of the excitation beam toward a photodiode, which converts the light to an electrical signal. This signal is used as a reference to compensate for intensity fluctuations during operation.

4. Light emitted by the sample in the flow cell is collected by the emission collector optics and is focused on the entrance slit of the emission monochromator. This diffracted emission spectrum is directed toward the photomultiplier tube, where it is amplified and converted into an electrical signal.

1

1.4.2 Control System

The control system consists of a microprocessor and drivers for controlling the components of the detector (Figure 1-3). The control system operates as follows:

1. Optical signals from the excitation and emission monochromators are converted into electrical signals by a photodiode and the photomultiplier tube, respectively. The electrical signals are then amplified by a preamplifier and converted to digital signals by an analog-to-digital (A/D) converter.

2. The microprocessor calculates the ratio of the two electrical signals to compensate for fluctuations in the intensity of the light source. The microprocessor then sends the resultant corrected signals to a digital-to-analog (D/A) converter for output on the analog connector.

3. The microprocessor receives and executes commands entered from the keypad or from the time program to control the operational parameters of the 474 detector, including the positions of the excitation and emission monochromators.

Figure 1-3 Block Diagram of the 474 Detector 1 Principle of Operation 31

Figure 1-3 Block Diagram of the 474 Detector 1 Principle of Operation 31

Figure 1-3 Block Diagram of the 474 Detector

1

2

Installing the 474 Detector

The Waters 474 Scanning Fluorescence Detector operates in any standard laboratory environment. The detector requires connection to electrical power and to sample and waste fluid lines.

This chapter describes how to install the 474 and connect it to the electrical and fluid supplies and to other equipment.

2.1 Site Selection and Power Requirements

Install the 474 at a site that meets the requirements listed in Table 2-1.

Table 2-1 Installation Site Requirements

2

Parameter

 

Requirement

Ambient temperature

10

to 32 °C (50 to 90 °F)

Relative humidity

20

to 80%, noncondensing

Bench space

5 inches (12.7 cm) clearance at rear

2 inches (5 cm) clearance on the sides

Vibration

Negligible

Static electricity

Negligible

Note: Be sure to provide at least 2 inches (5 cm) clearance for the vent on the side panel.

To prevent instability, make sure that the four rubber feet are secure on the bench top.

Power Requirements

The Waters 474 requires:

One properly grounded AC outlet

No nearby source of electronic noise

Correct amperage fuse for your AC voltage

Table 2-2 lists the fuse requirements for the two operating voltages.

2

Table 2-2 Voltage and Fuse Selection

Nominal Voltage

Voltage Range

Fuse Required

(VAC)

(VAC)

115

90 to 132

5 A, fast-blow

230

187 to 264

2.5 A, fast-blow

Caution: To avoid electric shock, power down the 474 and unplug the power cord before you To avoid electric shock, power down the 474 and unplug the power cord before you replace fuses.

Warning: To reduce the risk of fire hazard, always replace fuses with the same type and To reduce the risk of fire hazard, always replace fuses with the same type and rating.

2.2 Unpacking and Inspection

The 474 is packed and shipped in one carton that contains the following items:

Waters 474 Scanning Fluorescence Detector

Start-up kit

Validation certificate

Operators guide

Packing list

Unpacking

To unpack the 474:

1. Locate the packing list.

2. Unpack the contents of the shipping carton. As you unpack the carton, check the contents against the list to make sure you received all items.

3. Check the contents of the start-up kit against the list enclosed with the kit.

4. Save the shipping carton for future transport or shipment.

Inspection

If you see any damage or discrepancy when you inspect the contents of the carton, immediately contact the shipping agent and Waters Technical Service at (800) 252-4752, U.S. and Canadian customers only. Other customers, call your local Waters subsidiary or your local Waters Technical Service Representative, or call Waters corporate headquarters for assistance at (508) 478-2000 (U.S.).

2

Note: Make sure the serial number on the detector nameplate located on the side panel matches the number on the validation certificate.

For more information about the instrument warranty and serial number, refer to Appendix C, Warranty Information.

2.3

Voltage Selection and Fuse Installation

The voltage selection switch and fuse holders are located on the rear panel of the detector (Figure 2-1).

2

! UV - CAUTION - - ATTENTON - DISCONNECT POWER COUPER L'ALIMENTATION ALLOW COOLING TIME
!
UV
- CAUTION -
- ATTENTON -
DISCONNECT POWER
COUPER L'ALIMENTATION
ALLOW COOLING TIME
BEFORE SERVICING
LAISSER REFROIDIR
LA LAMPE AVANT
LAMP
DE TOUCHER
Fuse Holders
TP01143
Voltage Selection Switch

Figure 2-1 Voltage Selection Switch and Fuse Holders

Checking the Voltage Selector

Make sure that the voltage selection switch is properly set for your requirements.

selection switch is properly set for your requirements. Caution: To avoid electric shock, turn off the

Caution: To avoid electric shock, turn off the Power switch on the front panel, and disconnect the power cord before you change the voltage selector switch position or change a fuse.

With the rear panel of the detector facing you, check that the voltage selection switch is:

In the up (115 V) position for 100 to 120 VAC power

In the down (230 V) position for 220 to 240 VAC power

If the switch is in the wrong position, use a small, flat-blade screwdriver to move it to the correct position.

Installing Fuses

To install the fuses:

1. Use a small, flat-blade screwdriver to press in on the fuse holder and turn the fuse holder counterclockwise one-quarter turn. The spring-loaded fuse holder pops out when you withdraw the screwdriver.

2. Insert the fuse into the fuse holder. Make sure that the fuse is properly rated for your requirements. Refer to Table 2-2 to determine your fuse requirements.

3. Insert the fuse holder into the receptacle. Press in with the screwdriver and turn the holder until it is securely in place.

4. Repeat steps 1, 2, and 3 for the second fuse holder.

2.4 Making Fluid Connections

Before the initial start-up you must complete:

2

The fluid connections described in this section.

The electrical connections described in Section 2.5, Making Electrical Power Connections.

If you are using more than one detector in series in your HPLC system, Waters strongly recommends that you install the 474 as the last detector in the series. This avoids the higher pressures that can occur on the upstream side of the fluid pathway.

STOP
STOP

Attention: The pressure rating of the 474 flow cell is approximately 145 psi for the 16 µL cell (500 psi for the 5 µL cell). If this pressure is exceeded, the flow cell could be damaged. Do not connect any tubing or device that might cause backpressure to build.

The fluid connections to the 474 are located in the center of the front panel (Figure 2-2). To make the inlet and outlet tubing connections, you need to:

Cut the tubing

Attach the inlet and outlet compression fittings

Connect the tubing to the detector

2

Outlet Connection

™ MODE SIGNAL RESPONSE Waters 474 PRGM 1.0000 STD Scanning Fluorescence Detector 250 395 1000
MODE
SIGNAL
RESPONSE
Waters
474
PRGM
1.0000
STD
Scanning Fluorescence Detector
250
395
1000
256
EX λ
EM λ
GAIN
ATTEN
PROGRAM #
DIAG
Program
Scan
Program
Monitor
Run
ENERGY
LAMP OFF
FILTER
out
1
2
3
Auto
λ
Gain
Zero
16µL Flow Cell
AZ ENABLE
OFFSET
POLARITY
4
5
6
Atten
Resp
A.Z.
max pressure
1000 kPa (145 psi)
!
LAMP HRS
B/W
7
8
9
EM SP
EX SP
Slit
in
λ RANGE
0
End
Clear
Edit/Enter
TEMP
Chart
Shift
O
I
Marker
Inlet Connection
TP01141

Cutting Tubing

Figure 2-2 Fluid Connections

You need the following tools to cut the tubing:

File with cutting edge

Two cloth- or plastic-covered pliers

To cut the tubing:

1. Measure the length of tubing (1/16-inch O.D. stainless steel) you need to make the following connections:

Column to the detector inlet

Detector outlet to a suitable waste container

2. Use a file with a cutting edge to scribe the circumference of the tubing at the desired length.

3. Grasp the tubing on both sides of the scribe mark with cloth-covered pliers. Gently work the tubing back and forth until it separates.

4. File the ends smooth.

Assembling Fittings

To assemble each fitting:

1. Slide the compression screw over the tubing end, followed by the ferrule (Figure 2-3).

2. Mount the ferrule with its taper end facing the end of the tubing (Figure 2-3).

Compression Screw Ferrule Tube
Compression Screw
Ferrule
Tube

Tubing end (straight and smooth to achieve maximum column efficiency)

TP01139

Distance (determined by each application, such as union or column fitting)

Figure 2-3 Ferrule and Compression Screw Assembly

Making Connections

2

Make connections at the column outlet and detector inlet, and at the detector outlet by performing the following steps:

1. Seat each tubing end in the appropriate fitting.

2. Seat each ferrule by tightening the compression screw 3/4 turn past finger-tight.

Flushing the System

Since the Waters 474 is shipped dry, you must pump solvent through the unit before initial startup.

Flush the lines with filtered HPLC-grade methanol. Then pump your mobile phase, provided that there are no miscibility problems.

2.5

Making Electrical Power Connections

The two electrical connectors on the rear panel of the 474 are:

The AC input connector that provides the unit with the operating voltage

The analog connector that provides status information and data to auxiliary equipment

2

2.5.1 Making AC Power Connections

STOP
STOP

Attention: Be sure that the voltage selection switch on the rear panel is set correctly and the correct fuses are installed before applying AC power. Refer to Section 2.3, Voltage Selection and Fuse Installation, for details.

To connect the 474 to the AC power supply:

1. Plug the receptacle end of the power cord into the AC input connector on the rear panel of the detector (Figure 2-4).

2. Plug the other end of the power cord into a properly grounded AC power
2. Plug the other end of the power cord into a properly grounded AC power source.
!
UV
- CAUTION -
- ATTENTON -
DISCONNECT POWER
COUPER L'ALIMENTATION
ALLOW COOLING TIME
BEFORE SERVICING
LAISSER REFROIDIR
LA LAMPE AVANT
LAMP
DE TOUCHER
Analog Connector
AC Receptacle

TP01143

Figure 2-4 Rear Panel Electrical Connectors

2.6

Making Connections to Other Devices

You can connect the 474 to a wide range of HPLC system devices. This section describes how to connect the 474 to a:

Millennium Data System

Waters 2690 Separations Module

Waters 600 Series Controller

Waters 745/745B/746 Data Module

Chart Recorder

Waters 717plus Autosampler

Waters Carbamate System

Waters AccQTag System

Voltmeter

Required Materials

2

When connecting cables to the analog connector terminals, you need the following tools:

Small flat-blade screwdriver

Electrical insulation stripping tool

Cable Connecting Procedure

To connect cables from other HPLC system devices to the analog connector on the rear panel:

1. Strip 1/4 inch (5 mm) of insulation from the end of each wire.

2. Insert the bare wire into the appropriate terminal and secure it by tightening the screw.

3. Test the connection by gently pulling on the wire. The connection is secure if you cannot pull the wire out with a gentle tug.

2.6.1 Analog Connector Overview

The 474 is usually installed as an integral part of a data collection system. Although the 474 can be controlled manually via the keypad on the front panel, several functions can be controlled externally through connections to the analog connector on the rear panel (Figure 2-4). These functions are described in Section 3.6, Using External Functions.

2

STOP
STOP

Attention: To meet the regulatory requirements of immunity from external electrical disturbances that may affect the performance of this instrument, do not use cables longer than 9.8 feet (3 meters) when you make connections to the analog connector. Waters recommends a maximum cable length of 6.5 feet (2 meters). In addition, ensure that you always connect the shield of the cable to chassis ground at one instrument only.

The analog connector is a 15-pin terminal strip on the rear panel (Figure 2-5). This connector is used for analog data and signal output to other instruments, and analog signal input from other instruments. Figure 2-5 shows the pinout of this connector, and Table 2-3 describes the function of each terminal. Refer to Section 2.6, Making Connections to Other Devices, for information on connecting other instruments to the 474.

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

Figure 2-5 Analog Connector

Table 2-3 describes the terminals on the analog connector.

Table 2-3 Analog Connector Terminal Descriptions

Terminal

Function

Reference

Int (+ and )

Integrator output terminals. A 1 V full-scale unattenuated analog output signal appears on these terminals. This signal is unaffected by the setting in the Attenuation parameter.

Using Gain and Attenuationin Section 3.4.5, Attenuation

Rec (+ and )

Recorder output terminals. A 10 mV full-scale analog output signal appears on these terminals. The signal range is determined by the settings in the Gain and Attenuation parameters.

Using Gain and Attenuationin Section 3.4.5, Attenuation

Ex Out (+ and )

Excitation out output terminals. A 1 V full-scale analog signal that corresponds to the photodiode voltage. Use this signal to monitor the excitation energy during scans.

Section 3.6.1,

Excitation Energy

Out

Mark Out (+ and )

Marker output terminals. A momentary 1 to 3 mV marker signal appears on these terminals when either of the following occurs:

The Chart Mark key on the keypad is pressed

Section 3.6.2,

Mark Out

 

A contact closure signal occurs between the Mark In terminal and the () terminal

Cell Leak (+ and )

Output terminals to signal a fluid leak. When these contacts close, a leak has occurred inside the flow cell of the detector.

Section 3.6.3,

Cell Leak

Mark In (+)

Mark input terminal. A chart mark is added to the recorder output when a contact closure signal appears between this terminal and the () terminal.

Section 3.6.4,

Mark In

AZ In (+)

Auto Zero input terminal. The voltages on the Rec (+ and ) and Int (+ and ) terminals are set to zero when a contact closure signal appears between this terminal and the () terminal.

Section 3.6.5, Auto Zero In

2

Table 2-3 Analog Connector Terminal Descriptions (Continued)

2

Terminal

Function

Reference

Prgm Rst (+)

Time program input terminal. When a contact closure signal appears between this terminal and the () terminal, the currently selected program starts immediately. If a time program is running, the program stops, the initial conditions are reset, and the program starts again

Section 3.6.6,

Program Reset

2.6.2 Connecting the 474 to the Millennium Data System

The Millennium® Data System is a single or multi-system computer that performs data acquisition, processing, and management of chromatographic information.

The Millennium Data System includes a menu-driven software program specifically designed by Waters for chromatographers. Use the Millennium Data System to:

Acquire data

Process data

Generate and print reports

Store information (or data) in a central area and share this information with users who have proper security access

The connections between the 474 detector and the Millennium Data System vary depending on your version of the Millennium Data System.

2.6.2.1 Millennium Data System Version 3.05.01 or Lower

Millennium software version 3.05.01 requires that the detector analog signal be converted to a digital representation. Data acquisition and control with the Millennium Data System version 3.05.01 and lower requires connections between the Waters Bus SAT/IN module and the Laboratory Acquisition and Control/Environment (LAC/E) card.

Bus SAT/IN Module

The Waters Bus SAT/IN module (Figure 2-6) converts the analog signals from devices such as the 474 into digital form. It then transmits these digital signals to the Bus LAC/E card inside the Millennium Data System computer.

Figure 2-6 Bus SAT/IN Module (Front Panel)
Figure 2-6 Bus SAT/IN Module (Front Panel)

To connect the 474 to the Millennium Data System:

2

1. Connect the Bus SAT/IN module to the Bus LAC/E card in the Millennium Data System computer.

2. Connect the 474 to the Bus SAT/IN Module.

Attention: Do not power up the Bus SAT/IN module until you perform all procedures described in the Waters Bus SAT/IN Module Installation Guide. Improper powerup can Attention: damage the unit and void the warranty. damage the unit and void the warranty.

Attention: To avoid damaging the Bus SAT/IN module, always disconnect the power cord Attention: at either the wall outlet or the power supply before attaching or removing the power at either the wall outlet or the power supply before attaching or removing the power connection to the Bus SAT/IN module. The Bus SAT/IN module does not have a power switch.

Connecting the Bus SAT/IN Module to the Bus LAC/E Card

The Bus SAT/IN module connects to the Bus LAC/E card through an I/O Distribution Box (Figure 2-7).

To connect the Bus SAT/IN module to the Bus LAC/E card:

1. Connect the I/O Distribution Box to the 9-pin I/O distribution port on the Bus LAC/E card using the I/O distribution cable.

2. Connect the data terminal on the back of the Bus SAT/IN to Port 2 of the I/O Distribution Box using a serial cable.

3. Configure the serial port for the Bus SAT/IN module as described in the Millennium 2010 Chromatography Manager Software Users Guide, Chapter 4, Configuring a Chromatographic System.

2

Guide , Chapter 4, Configuring a Chromatographic System. 2 Figure 2-7 Bus SAT/IN to Bus LAC/E
Guide , Chapter 4, Configuring a Chromatographic System. 2 Figure 2-7 Bus SAT/IN to Bus LAC/E
Guide , Chapter 4, Configuring a Chromatographic System. 2 Figure 2-7 Bus SAT/IN to Bus LAC/E

Figure 2-7 Bus SAT/IN to Bus LAC/E Connections

Connecting the 474 to the Bus SAT/IN Module

The 474 connects to the Bus SAT/IN module as shown in Figure 2-8.

Waters 474

Analog Connector

Figure 2-8 Bus SAT/IN Module Connections
Figure 2-8 Bus SAT/IN Module Connections
15 + Int 14 − 13 12 + Rec − 11 10 + Ex Out
15
+
Int
14
13
12
+
Rec
11
10
+
Ex Out
9
8
+
Mark Out
7
6
+
Cell Leak
5
4
+
Mark In
3
+
AZ In
2
+
Prgm Rst
1

To connect the 474 to the Bus SAT/IN module:

2

1. Connect the analog cable (included with the Bus SAT/IN module) to the Int (+ and ) terminals on the rear panel of the 474.

2. Connect the other end of the cable to either the Channel 1 or Channel 2 connector on the front panel of the Bus SAT/IN module.

The 474-to-Bus SAT/IN connections are summarized in the following table.

474 Analog Connector Terminal

Bus SAT/IN

Connector

Int (+)

+ Lead from Channel 1 or 2

Int ()

Lead from Channel 1 or 2

2.6.2.2 Millennium 32 Data System Version 3.2 or Higher

Before you connect the 474 to the Millennium 32 Data System version 3.2 or higher, ensure that:

The Windows® NT operating system is installed in the Millennium 32 computer

The 474 detector is equipped with the RS-232 Control Option Kit (Part Number

2

176000204)

For detectors with a serial number:

B00474712M or higher, the RS-232 Control Option kit is factory installed.

B00474711M or lower, the RS-232 Control Option kit is installed on site by a Waters Field Service Representative.

See the Waters 474 Scanning Fluorescence Detector RS-232 Control Option Guide for information about installing the RS-232 Control Option Kit.

To connect the 474 to the Millennium 32 Data System:

1. Connect the 25-pin end of the null modem cable (included in the RS-232 Control Option Kit) to the RS-232 port on the rear panel of the 474.

2. Connect the other end of the cable to one of the COM ports on the rear panel of the Millennium 32 computer.

Note: If your Millennium 32 computer does not have an available COM port, install the optional eight-port serial card in the Millennium 32 computer. Use one of the cables provided with the card along with a 25-pin adapter (part number 340000103) to connect the 474 to one of the serial ports. For more information about using the eight-port serial card, see the Millennium 32 Installation and Configuration Guide, section 2.3, Connecting Chromatographic Devices.

To configure the 474 detector address on the eight-port serial card, use the Acquisition Server Properties window in the Millennium 32 Configuration Manager. For more information about configuring the serial ports, see the Configuring Serial Ports topic in the Millennium 32 Online Help.

3. Use a signal cable to make the connections between the 474 and a Waters 2690 Separations Module or a Waters 600 Series Controller that are indicated in the following table.

474 Analog Connector Terminal

Waters 2690 Connector B Terminal or Waters 600 Series Controller Terminal

Mark In (+)

Inject Start

Mark In (+) Inject Start Inject Start

Inject Start

2.6.3 Connecting the 474 to the Waters 2690 Separations Module

To connect a standalone Waters 2690 Separations Module to the 474, you must make the following connections:

Auto Zero

Chart Mark

Method Start

Cell Leak

2

If your 2690 Separations Module is part of a Carbamate Analysis System or an AccQTag

system, see Section 2.6.8, Connecting the 474 to the Carbamate System or Section 2.6.9, Connecting the 474 to the AccQTag System.

Auto Zero

A contact closure signal between the AZ In (+) and the () terminals of the 474 starts the

auto zero function. This function sets the analog voltage to zero on the Rec (+ and ) and Int (+ and ) terminals of the 474.

To provide an auto zero signal to the 474, use a signal cable to make the connections between the 474 and a Waters 2690 Separations Module as summarized in the following table, and as illustrated in Figure 2-9.

474 Analog Connector Terminal

Waters 2690 Connector B Terminal

AZ In (+)

Inject Start

Inject Start

Inject Start

2

Waters 2690 Waters 474 Connector B Analog Connector 15 + Int 14 − 1 13
Waters 2690
Waters 474
Connector B
Analog Connector
15
+
Int
14
1
13
Inject Start
12
+
2
Rec
11
Ground
3
10
+
+
4
Ex Out
Stop Flow
9
5
8
+
Mark Out
+
6
7
Hold Inject 1
7
6
+
Cell Leak
+
8
5
Hold Inject 2
9
4
+
Mark In
Ground
10
3
+
AZ In
+ 11
2
+
Prgm Rst
Chart Out
− 12
1

Figure 2-9 Waters 2690 Separations Module Auto Zero Connections

Chart Mark

A contact closure signal between the Mark In (+) and the terminals of the 474 creates a chart mark signal, which is a momentary 1- to 3-mV increase in the analog voltage on the Rec (+ and ) and Mark Out (+ and ) terminals. The chart mark can signal events such as the start of an injection.

To provide a chart mark signal from a Waters 2690 Separations Module to the 474:

1. Use a signal cable to make the connections between the 474 and a Waters 2690 Separations Module as summarized in the following table, and as illustrated in Figure 2-10.

474 Analog Connector Terminal

Waters 2690 Connector A Terminal

Mark In (+)

Switch 1, 2, 3, or 4 (+)

Switch 1, 2, 3, or 4 ( – )

Switch 1, 2, 3, or 4 ()

2. Program the 2690 Separations Module to provide a pulse output on the switch at the beginning of each run. Refer to the Waters 2690 Separations Module Operators Guide, Section 2.7.1, I/O Signal Connections, for more details.

Waters 474

Waters 2690 Analog Connector Connector A 15 + Int 14 − 13 1 12 +
Waters 2690
Analog Connector
Connector A
15
+
Int
14
13
1
12
+
Switch 1
Rec
2
11
Ground
3
10
+
Ex Out
4
9
Switch 2
5
8
+
Mark Out
6
7
Switch 3
6
+
7
Cell Leak
5
8
Switch 4
4
+
Mark In
9
3
+
AZ In
Ground
10
2
+
Prgm Rst
11
Run Stopped
1
12

2

Figure 2-10 Waters 2690 Separations Module Chart Mark Connections

Refer to Section 3.6.4, Mark In, for a complete description of the Mark In terminal.

Method Start

To provide a method start signal to the 474, use a signal cable to make the connections between the 474 and a Waters 2690 Separations Module as summarized in the following table, and as illustrated in Figure 2-11.

2

474 Analog Connector Terminal

Waters 2690 Connector B Terminal

Prgm Rst (+)

Inject Start (+)

Inject Start ( – )

Inject Start ()

Waters 2690

Connector B

Waters 474

Analog Connector

1 Inject Start 2 Ground 3 + 4 Stop Flow − 5 + 6 Hold
1
Inject Start
2
Ground
3
+
4
Stop Flow
5
+
6
Hold Inject 1
7
+
8
Hold Inject 2
9
Ground
10
+ 11
Chart Out
− 12

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

Figure 2-11 Waters 2690 Separations Module Method Start Connections

Cell Leak

The 474 generates a contact closure signal when a leak occurs in the flow cell cassette. You can use this signal as an alarm signal to the 2690 Separations Module.

To provide a signal for an external warning when a leak occurs in the flow cell, make the connections summarized in the following table, and as illustrated in Figure 2-12.

474 Analog Connector Terminal

Waters 2690 Connector B Terminal

Cell Leak (+)

Stop Flow (+)

Cell Leak ()

Stop Flow ()

Waters 2690

Connector B

Waters 474

Analog Connector

1 Inject Start 2 Ground 3 + 4 Stop Flow − 5 + 6 Hold
1
Inject Start
2
Ground
3
+
4
Stop Flow
5
+
6
Hold Inject 1
7
+
8
Hold Inject 2
9
Ground
10
+ 11
Chart Out
− 12

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

2

Figure 2-12 Waters 2690 Separations Module Cell Leak Connections

2.6.4 Connecting the 474 to the Waters 600 Series Controller

To connect a standalone Waters 600 series controller to the 474, you must make the following connections:

2

Auto Zero

Chart Mark

Method Start

Cell Leak

Auto Zero

A contact closure signal between the AZ In (+) and the () terminals of the 474 starts the auto zero function. This function sets the analog voltage to zero on the Rec (+ and ) and Int (+ and ) terminals of the 474.

To provide an auto zero signal to the 474, use a signal cable to make the connections between the 474 and a Waters 600 series controller as summarized in the following table, and as illustrated in Figure 2-13.

474 Analog Connector Terminal

Waters 600 Series Controller Terminal

AZ In (+)

Inject

474 Analog Connector Terminal Waters 600 Series Controller Terminal AZ In (+) Inject
474 Analog Connector Terminal Waters 600 Series Controller Terminal AZ In (+) Inject

Waters 474

Analog Connector

Waters 600 Series Controller
Waters 600 Series Controller

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

2

Figure 2-13 Waters 600 Series Controller Auto Zero Connections

Chart Mark

A contact closure signal between the Mark In (+) and the terminals of the 474 creates a chart mark signal, which is a momentary 1- to 3-mV increase in the analog voltage on the Rec (+ and ) and Mark Out (+ and ) terminals. The chart mark can signal events such as the start of an injection.

To provide a chart mark signal from a Waters 600 series controller to the 474:

1. Use a signal cable to make the connections between the 474 and a Waters 600 series controller as summarized in the following table, and as illustrated in Figure 2-14.

474 Analog Connector Terminal

Waters 600 Series Controller Terminal

Mark In (+)

S1, S2, S3, or S4

GND

GND

2

Waters 474

Analog Connector

Waters 600 Series Controller
Waters 600 Series Controller

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

Figure 2-14 Waters 600 Series Controller Chart Mark Connections

2. Program the 600 controller to provide a pulse output on the switch at the beginning of each run. Refer to the Waters 600E Multisolvent Delivery System Users Guide, Section 5.1.2, for more details.

For a complete description of the Mark In terminal, refer to Section 3.6.4, Mark In.

Method Start

To provide a method start signal to the 474, use a signal cable to make the connections between the 474 and a Waters 600 series controller as summarized in the following table, and as illustrated in Figure 2-15.

474 Analog Connector Terminal

Waters 600 Series Controller Terminal

Prgm Rst (+)

Inject (+)

474 Analog Connector Terminal Waters 600 Series Controller Terminal Prgm Rst (+) Inject (+)
474 Analog Connector Terminal Waters 600 Series Controller Terminal Prgm Rst (+) Inject (+)

Waters 474

Analog Connector

Waters 600 Series Controller
Waters 600 Series Controller

15

+

Ex Out

Mark Out

Cell Leak

14

13

12

+

11

10

+

 

9

8

+

 

7

6

+

 

5

4

+

Mark In

3

+

AZ In

2

+

Prgm Rst

1

1

Figure 2-15 Waters 600 Series Controller Connections

2

Cell Leak

The 474 generates a contact closure signal when a leak occurs in the flow cell cassette. You can use this signal as an alarm signal to the 600 Series Controller.

To provide a signal for an external warning when a leak occurs in the flow cell, make the connections summarized in the following table and illustrted in Figure 2-16.

2

474 Analog

Waters 600 Series Controller Terminal

Connector

Cell Leak (+)

Stop Flow

Cell Leak ()

GND

Waters 474

Analog Connector

Waters 600 Series Controller
Waters 600 Series Controller
15 + Int 14 − 13 12 + Rec − 11 10 + Ex Out
15
+
Int
14
13
12
+
Rec
11
10
+
Ex Out
9
8
+
Mark Out
7
6
+
Cell Leak
5
4
+
Mark In
3
+
AZ In
2
+